PIWI‐Interacting RNA HAAPIR Regulates Cardiomyocyte Death After Myocardial Infarction by Promoting NAT10‐Mediated ac4C Acetylation of Tfec mRNA

Abstract PIWI‐interacting RNAs (piRNAs) are abundantly expressed in heart. However, their functions and molecular mechanisms during myocardial infarction remain unknown. Here, a heart‐apoptosis‐associated piRNA (HAAPIR), which regulates cardiomyocyte apoptosis by targeting N‐acetyltransferase 10 (NAT10)‐mediated N4‐acetylcytidine (ac4C) acetylation of transcription factor EC (Tfec) mRNA transcript, is identified. HAAPIR deletion attenuates ischemia/reperfusion induced myocardial infarction and ameliorate cardiac function compared to WT mice. Mechanistically, HAAPIR directly interacts with NAT10 and enhances ac4C acetylation of Tfec mRNA transcript, which increases Tfec expression. TFEC can further upregulate the transcription of BCL2‐interacting killer (Bik), a pro‐apoptotic factor, which results in the accumulation of Bik and progression of cardiomyocyte apoptosis. The findings reveal that piRNA‐mediated ac4C acetylation mechanism is involved in the regulation of cardiomyocyte apoptosis. HAAPIR‐NAT10‐TFEC‐BIK signaling axis can be potential target for the reduction of myocardial injury caused by cardiomyocyte apoptosis in ischemia heart diseases.


Animal experiments and Generation of HAAPIR knockout (HAAPIR KO) mice.
Adult male mice of 8-10 weeks were used in this study. Mice were raised at 24±2℃ with an interval of 12 hours for a dark or light cycle. All animal experiments were conducted according to the protocols approved by Laboratory Animal Welfare Ethics Committee of Qingdao University, Qingdao, China.
HAAPIR knockout mice were generated by using clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas9) gene-editing system (Cyagen Biosciences Inc. Guangzhou, China). HAAPIR +/-mice were interbred to generate KO mice (HAAPIR -/-), which were used for further studies. Mice were genotyped by PCR with forward primer: GACCAACCCCAACAGTCTCTCATC and reverse primer: TCCGTGGCTGTCTTATCATGGATC. The PCR products were further sequenced to verify that the HAAPIR was correctly deleted. All experiments were performed on HAAPIR-/-mice and their WT littermates. I/R injury. For the establishment of I/R injury, the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (10ml kg-1 mouse). Under aseptic conditions, the heart was exposed by left thoracotomy, and the left anterior descending (LAD) coronary artery was ligated in the temporal region for 45 min of ischemia and followed by 3 h reperfusion as previously described 1 . In the Sham-operated group, a similar procedure except the snare was left untied. For Tfec knockdown in vivo, the Tfec knockdown vector was constructed by recombinant serum type 9 adeno associated virus system (AAV9) (Hanbio, Shanghai, China). Mice were given tail vein injection 3 weeks before Sham or I/R operation (5×10 10 vg/mouse). The hearts were collected in the in-vivo study after 45mins ischemia and 3 hours reperfusion.
Echocardiography measurement and Myocardial infarct size measurement. After I/R or Sham operation, echocardiographic measurements were performed using the Vevo2100 imaging system (Visual Sonic) to assess cardiac function. After echocardiography, the mice were sacrificed and the hearts were removed for further study. After echocardiography, 1% Evans blue dye (Sigma, St. Louis, Missouri, USA) was injected into jugular vein of the mice, and the hearts were removed and transversely sectioned serially into 2 mm-thick slices. Then, the heart slices were incubated in 1.5% 2,3,5-triphenyltetrazolium chloride (Sigma, St. Louis, Missouri, USA) at 37℃ for 10-30 min under dark conditions. After removal, PBS was washed for 5 min. 4% paraformaldehyde (Servicebio, G1101, Wuhan, China) was fixed at room temperature for 15-25min, and the infarcted area was taken by camera (Nikon-D5600) and analyzed by ImageJ 7.0 (NIH, Boston, USA).
Transfection of HAAPIR agomir and antagomir. All HAAPIR agomir or antagomir and their negative controls were obtained from Gene Pharma (Shanghai, China). Agomir or antagomir of HAAPIR was transfected into cardiomyocytes using Lipofectamine 3000 (Invitrogen, NY, USA). Taking 6-well plate as an example.
Specifically, mix 5μl of Lipofectamine 3000 and an equal volume of solution containing agomir or antagomir of HAAPIR with 125 μl of serum-free DMEM/F12, respectively, and let stand for 5 min, then mix the two. Let stand for 15-20 min and add to a well in a 6-well plate containing 2ml DMEM/F12 medium. The HAAPIR agomir sequence is 5'-UCCAUUCACUCCUUCCUUCCCGACUAUUGG-3', the agomir negative control (agomir-NC) sequence is The antagomir sequence is 5'-CCAAUAGUCGGGAAGGAAGGAGUGAAUGGA-3', the antagomir negative control (anta-NC) sequence is 5'-CAGUAUUUUGUGUAGUACAA-3'. at room temperature for 10-30min under dark conditions. After washing, the cells were anti-stained with DAPI and observed under fluorescence microscope. Then, the PI-positive nuclei were counted and the total of PI-positive cells were expressed as a percentage of total DAPI-positive nuclei (represents the total number of the cells).

RNA interference (RNAi
The LDH assay kit (Jiancheng, Nanjing, Jiangsu, China) was used to determine the activity of LDH released from cell culture medium according to the instructions. After the collected cell culture medium were treated according to the instructions, the absorbance was measured at 450nm wavelength by enzyme labeling instrument (BioTek, Synergy Neo2, USA), and the LDH activity was calculated.
Nuclear and cytoplasmic RNA extraction. Nuclear/Cytosol fractionation kit (Biovision, Inc, CA) was used to separate cytoplasmic and nuclear extracts from cardiomyocytes. According to the instructions, the myocardial myocytes were lysed with cytosol extraction buffer A (CEB-A) and CEB-B at low temperature, centrifuged at 16000g, and the supernatant was used as cytoplasmic extract. After the precipitation was suspended with nuclear extraction buffer (NEB), the supernatant was used as nuclear extract after vortex centrifugation. Then the RNA was extracted using Trizol (Sigma, St. Louis, Missouri, USA).

RNA pulldown and RNA-binding protein immunoprecipitation (RIP) assay.
Cardiomyocytes transfected with biotinylated HAAPIR and NC (Gene Pharma, Shanghai, China) were collected, and briefly incubated in lysate buffer containing protease inhibitor. The lysate mixture (10% of total volume) was taken as input and the rest of the mixture was incubated with streptavidin-sepharose beads. Then the beads were cleaned and boiled with loading buffer, and immunoblot was performed.
RIP assay was performed using the Megna RIP RNA-binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer's instructions. In brief, the cells were lysed with RIP lysis buffer (50mM Tris pH 8.0, 150mM NaCl, 10% Glycerol, 1mM EDTA, 50mM NaF, 0.1% NP-40) containing protease and RNAase inhibitors. and the whole lysate (100 l) incubated overnight with the primary antibodies or IgG (negative control) coated beads at 4°C. The protein-RNA complexes bound with beads were captured with magnetic protein A/G beads and centrifuged at 12000×g for 5 min at 4°C. The supernatant was discarded, RNA-binding protein were washed, eluted and treated with Proteinase K. The purified RNA was extracted with Trizol reagent and qRT-PCR was conducted.  The experiments were repeated independently at least three times with similar results and p < 0.05 was considered statistically significant.